Botulinum toxin (BoNT) is a microbial protein that causes a potentially fatal neuroparalytic disease called botulism. The disease can occur in several different variants, but the most common is oral poisoning. Patients can ingest food that is contaminated with preformed toxin (primary infection), or they can ingest food contaminated with organisms that manufacture toxin in situ (primary infection with secondary intoxication). In both cases, the toxin progresses through two essential sequences of events to produce its adverse effects.
During the first sequence of events, BoNT is absorbed into the body. More precisely, the toxin binds to the apical surface of epithelial cells in the gut (viz., transport cells). This is followed by receptor-mediated endocytosis, transcytosis, and eventual release of unmodified toxin into the general circulation. The toxin is distributed throughout the periphery, where it binds with high affinity to the junctional region of cholinergic nerve endings (viz., target cells). This initiates the second sequence of events, which includes receptor-mediated endocytosis, pH-induced translocation to the cytosol, and enzymatic cleavage of polypeptides that govern transmitter release. Cleavage of these substrates, with the resulting blockade in transmitter exocytosis, produces the neuroparalytic outcome that is characteristic of the disease botulism.
BoNT is synthesized as a single-chain inactive propolypeptide having a molecular mass of approximately 150 kilodaltons. Inactive pro-BoNT is activated by proteolytic cleavage of the pro-BoNT by endogenous or exogenous proteases. Cleavage (“nicking”) of the inactive BoNT propeptide yields two polypeptide chains, a heavy chain (“HC”) and a light chain (“LC”). The HC and LC normally remain linked by a disulfide bond that can be severed under reducing conditions, such as those that exist in the interior of an animal cell.
Recent studies have demonstrated that: (a) the BoNT molecule possesses the ligand properties that account for binding to epithelial cells; auxiliary proteins that are associated with the toxin are not essential for binding; (b) the entire light chain and the aminoterminal portion of the heavy chain can be removed from the holotoxin, and the residual about 50 kDa carboxyterminal portion of the heavy chain (HC50) retains the ability to cross epithelial barriers, and (c) both the holotoxin and the HC50 domain cross epithelial monolayers without any change in their characteristic structures and biological activities (Maksymowych et al., Infect. Immun. 67:4708-4712, 1999; Maksymowych et al., J. Biol. Chem. 273:21950-21957, 1998; Maksymowych et al., J. Pharmacol. Exp. Ther. 310:633-641, 2004). Thus, the BoNT heavy chain, and the HC50 domain in particular, have been proposed as an antigen in developing a mucosal vaccine against botulinum toxin and also as a delivery vehicle in transporting entities across epithelial barrier. See US Patent Publication 2004/0013687 A1 (2004).
The three-dimensional structure of BoNT serotype A (BoNT/A) has been determined (Lacy et al., Nature Struct. Biol. 5:898-902, 1998; Lacy et al., J. Mol. Biol. 291:1091-1104, 1999). The toxin molecule is composed of three somewhat independent lobes that represent the light chain (ca. 50 kDa), the aminoterminal portion of the heavy chain (ca. 50 kDa), and the carboxyterminal portion of the heavy chain (ca. 50 kDa). It is the third lobe that plays a key role in binding to nerve terminals, and it is this portion of the molecule that displays affinity for gangliosides. Certain single point mutations in the BoNT/A carboxyterminal portion have been shown to diminish, but not completely abrogate, binding to nerve-membrane preparations (Rummel et al., Mol. Microbiol. 51:631-643, 2004).
When administered by injection, BoNT HC50 polypeptides evoke a robust IgG response (Byrne et al., Biochimie 82:955-966, 2000). While BoNT/A HC50 evokes an immune response, it also carries a potential risk. The HC50 domain can bind and enter nerve endings (Lalli et al., J. Cell Sci. 112:2715-2724, 1999). Clearly, this is not a desirable characteristic of a vaccine candidate, nor of a delivery vehicle for transporting chemical entities across epithelial membranes. Therefore, alterations in the HC50 domain of BoNT/A HC polypeptides that abolish neuronal binding but that do not abolish epithelial transport or immunogenicity would represent a substantial advance.
What is needed is a BoNT-based polypeptide, particularly a BoNT/A-based polypeptide, which may be safely employed as a botulinum vaccine or a transmucosal carrier for delivery of therapeutic or diagnostic agents to mammals, without neurotoxicity to the subject. In particular, what is needed is a BoNT-based polypeptide that has been engineered to lose its affinity for polysialogangliosides and nerve endings, but retains its affinity for epithelial cells.